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Biomedical subjects

C A Walter

Publications and source records attributed to C A Walter.

17 recordsLinked to original sources
PubMed1992

Expression of a human chimeric transferrin gene in senescent transgenic mice reflects the decrease of transferrin levels in aging humans.

Transgenic mice provide a means to study human gene expression in vivo throughout the aging process. A DNA sequence containing 668 bp of the 5' regulatory region of the human transferrin gene was fused to the bacterial reporter gene chloramphenicol acetyl transferase (TF-CAT) and introduced into the mouse genome. Expression of the human chimeric transferrin gene was similar to the tissue patterns of mouse and human transferrin. In aging transgenic mice, expression of the human chimeric transferrin gene was found to diminish 40% in livers between 18 and 26 months of age. Transferrin levels and serum iron levels in aging humans also diminish, as observed from measurements of total iron binding capacity and percent iron saturation in sera from 701 individuals ranging from 0 to 99 years of age. In contrast, in transgenic mice and nontransgenic mice, the mouse endogenous plasma transferrin and endogenous Tf mRNA increase significantly during aging. Neither the decrease of human TF-CAT nor the increase of mouse transferrin during aging appears to be part of a typical inflammatory reaction. Although the 5' regions of the human transferrin and mouse transferrin genes are homologous, sequence diversities exist which could account for the different responses to inflammation and aging observed.

Adolescent↗
PubMed1991

Characterization of Chinese hamster ovary cells stably transformed by a plasmid with an inducible APRT gene.

A plasmid was constructed by fusion of a selectable mammalian gene, hamster adenine phosphoribosyltransferase (APRT), to the Zn(2+)-inducible sheep metallothionein I (MT I) promoter. This plasmid was used to produce stable Chinese hamster ovary (CHO) cell transformants by electroporation to study the effects of induced gene expression on DNA-mediated transformation. The sheep MT Ia promoter was chosen for these experiments because it regulates gene expression differently than murine MT promoters, exhibiting low basal levels of gene expression in uninduced conditions. We have shown that in the absence of Zn2+, there is very low expression of a sheep MT I-APRT fusion gene in stable CHO cells transformants; induction of APRT mRNA and enzyme activity by Zn2+ produced a "threshold" response, from low basal levels to high induced levels, in Zn2+ responsive stable transformant clones. In electroporation experiments, transformation frequencies were unaffected by Zn2+ treatments during a preselection period, but the presence of Zn2+ during selection increased the recovery of stable transformant clones 8- to 10-fold. All stable transformants analyzed displayed Zn(2+)-inducible APRT enzyme activity. Our results indicate that stable mammalian cell transformants with inducible genes under regulation of the sheep MT I promoter should be useful, because of low basal and high induced expression, for studies in which modulation of transcriptional activity is required.

Adenine Phosphoribosyltransferase↗
PubMed1990

Human transferrin. Expression and iron modulation of chimeric genes in transgenic mice.

Transferrin (TF) is a plasma protein that transports and is regulated by iron. The aim of this study was to characterize human TF gene sequences that respond in vivo to cellular signals affecting expression in various tissues and to iron administration. Chimeric genes were constructed containing 152, 622, and 1152 base pairs (bp) of the human TF5'-flanking region with the coding region of a reporter gene, CAT (chloramphenicol acetyltransferase), and introduced into the germ line of mice. Transgenes containing TF 5'-flanking sequences to -152 bp were expressed poorly in all tissues examined. In contrast, transgenes containing TF sequences to -622 or -1152 bp were expressed at high levels in brain and liver, greater than or equal to 1000-fold higher than tissues such as heart and testes. Liver and brain are major sites of endogenous TF mRNA synthesis, but liver mRNA levels are 10-fold higher than brain. A significant diminution of CAT enzymatic activity in liver accompanied iron administration in both TF(0.67) and TF(1.2)CAT transgenic mice, mimicking the decrease of transferrin in humans following iron overload. Levels of endogenous plasma transferrin also decreased in iron-treated transgenic mice. Transgenic mouse lines carrying human TF chimeric genes will be useful models for analyzing the regulation of human transferrin by iron and for determining the molecular basis of transferrin regulation throughout mammalian development into the aging process.

Animals↗
PubMed1978

Oncornavirus lytic activity in the serum of gibbon apes.

Fresh blood serum from normal gibbon apes (Hylobates lar) contained heat-sensitive lytic activity for various mammalian oncornaviruses. Lytic activity quantitatively similar to that in gibbon serum was demonstrated in serum from three other primate species, including man; it was demonstrated to be low or absent in lower mammalian species with the exception of domestic cats, which had intermediate levels of serum lytic activity. Gibbons that acquired infectious gibbon ape leukemia virus, either naturally by exposure to a virus-shedding ape or experimentally by deliberate virus inoculation, had the same levels of serum lytic activity as did unexposed gibbons that had no detectable antibodies to gibbon ape leukemia virus. A leukemic-viremic gibbon had low or absent serum oncornavirus lytic activity. These results indicated that serum lytic activity does not necessarily protect against infection by oncornaviruses, although it may limit virus replication and/or dissemination.

Animals↗
PubMed1977

Hypothermic perfusion of rabbit kidneys with solutions containing gelatin polypeptides.

Rabbit kidneys were perfused with a solution of extracellular electrolyte composition, made hypertonic with glucose and containing the gelatin polypeptide preparation Haemaccel (Hoechst) as the only colloid. Perfusions were carried out at 5 and 10 C for 19 hr, and function was tested by autografting. All of the kidneys perfused at the higher temperature showed immediate life-sustaining function after transplantation and contralateral nephrectomy, whereas only one graft of five perfused at the lower temperature showed any function. The suitability of the Haemaccel solution as a vehicle for introducing the cryoprotective agent glycerol was tested by perfusing kidneys for 4 hr with a solutiont containing 2% glycerol; the function of these organs was similar to that of kidneys transplanted without perfusion. Ultrastructural examination of kidneys perfused for 24 hr at 10 C showed excellent structural preservation, but measurements of water and ion contents and the penetration of marker molecules in nonmetabolizing kidneys showed 2.8% Haemaccel to be somewhat less effective than 6% bovine serum albumin in stabilizing these values. The Haemaccel perfusate is considered to be highly suitable for the introduction and removal of cryoprotective agents, and the results of hypothermic preservation by continuous perfusion are encouraging.

Animals↗
PubMed1977

Structural and functional aspects of biological freezing techniques.

The cooling procedures used to prepare samples for ultrastructural examination at low temperatures often differ markedly from those used to recover optimal function of cells on thawing. The implications of these differences are reviewed. Damage and alteration to the structure and function of the cells may be caused by the high concentrations of cryoprotective agents such as glycerol or dimethyl sulphoxide (DMSO) often added to reduce ice crystal artefacts. Under the rapid cooling conditions commonly employed for structural studies, these additives are not cryoprotective; low rates of cooling are necessary for them to be effective. Rapidly cooled cells that contain intracellular ice are only injured during rewarming so their structure may be as yet unaltered by any damaging effects at low temperatures. Most cells able to recover on thawing are grossly shrunken at low temperatures but since they are potentially functional they are of interest structurally. These cryobiological principles are illustrated with freeze-fracture, freeze substitution and functional assays. The cell types chosen were Chlorella sp. and mammalian tissue culture cells.

Animals↗
PubMed1977

Preservation of intraerythrocytic forms of malarial parasites by one-step and two-step cooling procedures.

Ring, trophozoite, and schizont stages of Plasmodium knowlesi were cooled in dimethyl sulfoxide either by direct immersion in liquid nitrogen or by a two-step method in which the cells were held at temperatures slightly below 0 degrees C for different lengths of time before they were cooled to -196 degrees C. After the direct plunge treatment, thawed trophozoites and schizonts were found to be extensively damaged. Their survival was markedly increased by holding them at -31 degrees C for 30 min before plunging them into liquid nitrogen. Freeze-substitution showed that cells cooled by the two-step procedure were grossly shrunken and had relatively few intracellular ice cavities. Large amounts of ice formed in trophozoites and schizonts preserved by direct immersion in liquid nitrogen. The two-step protocols investigated did not improve the survival of ring-stage parasites, 25-50% of which survived rapid cooling to -196 degrees C. Infected cell agglutination tests were carried out with frozen and thawed schizonts. Variant specificity was demonstrated with cells that had been plunged directly to -196 degrees C, but cells cooled by the two-step method tended to agglutinate spontaneously.

Animals↗
PubMedDate not supplied

The cryobiological basis for cryosurgery.

This paper discusses two important biological factors contributing to the death of cells within any region that is frozen cryosurgically. The first factor is the relationship between cell death and the thermal history experienced by each cell. Freezing with a single probe as usually done cannot provide sufficiently uniform conditions of cooling and warming to ensure widespread cell death. This situation can be improved by cycling a single probe at subzero temperatures or by the use of a multiple probe. The second factor is that of possible immunological responses induced by cryosurgical freezing. Improvements in understanding of biological implications of low temperatures in cryosurgery will aid the practical usefulness of these techniques.

Animals↗